Sol-gel monolithic column with optical window and method of making

ABSTRACT

A method of preparing a sol-gel monolithic column includes the step of forming a separation bed ( 14 ) from a sol-gel solution in a single process step. This column has improved characteristics for CEC based on its incorporated surface charge and ease of operation due to a lack of or need for end frits. Also, a second type of column includes an optical window ( 30 ) for on-column detection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a National Phase Concerning a Filing Under 35U.S.C. 371, claiming the benefit of priority of PCT/US01/04271, filed 9Feb. 2001, which claims the benefit of priority of U.S. ProvisionalApplication Ser. No. 60/181,371, filed 9 Feb. 2000 and U.S. ProvisionalApplication Ser. No. 60/181,642, filed 10 Feb. 2000, all of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to columns and methods of making columnsfor separation techniques and apparatus. More specifically, the presentinvention provides a separation bed and method of making the same foruse in various electromigration and non-electromigration separationcolumns, such as high-performance liquid chromatography, gaschromatography, capillary electrophoresis, capillaryelectrochromatography, and supercritical fluid chromatography.

2. Description of Related Art

Capillary electrochromatography or CEC is a fairly novel electrokineticseparation technique representing a hybrid of high-performance liquidchromatography or HPLC and capillary electrophoresis, known as CE. InCEC, the electroosmotic flow, or EOF is used to drive the mobile phasethrough the capillary, using typical HPLC mobile and stationary phasesthat provide the essential chromatographic interactions. Because of theflat plug-like profile of the electroosmotic flow, CEC offers greatlyenhanced separation efficiencies relative to HPLC. Unlike CE, CEC is notrestricted to charged solutes. Thus, the potential for CEC, as aseparation technique, is much wider.

Capillary electrochromatography is a rapidly growing area in analyticalseparations. A great deal of research effort is currently being devotedto materialize the great analytical potential that this new hybridtechnique has to offer. In order for CEC to achieve success as anindependent chromatographic separation technique significantadvancements are needed in the area of column technology. This isexplained by the fact that in CEC, the column not only serves as theseparation chamber, but also as the pumping device to drive the mobilephase through the system. This makes the column the “heart” of the CECsystem both in the functional and literal sense of the word.

Two major types of columns are used in current CEC practice. These arepacked and open tubular types. Packed columns comprise the predominantclass of CEC columns. Most often the packed capillaries contain 1.5-5μm, non-polar, octadecylated or ODS particles. The ODS particles possessboth the chemically bonded octadecyl stationary phase, providing theessential chromatographic interactions, and the silanol moieties,responsible for the generation of electroosmotic flow to drive themobile phase and the solutes through the packed capillary. Thecommercial availability of the ODS-bonded particles and the previouslyestablished liquid chromatography or LC separation protocols are twoadvantages attracting many researchers to use these packed capillariesin CEC. However, the most significant advantage of packed columns in CECis the possibility of using small micrometer and nanometer sizeparticles. High separation efficiency during fast analysis is achievedin packed CEC columns without requiring ultra-high pressures, as in HPLCto drive the mobile phase through the columns packed with the smallparticles.

The greatest challenge is the preparation of a uniform packing bed usingthe small particles. Researchers currently use a variety of packingprocedures ranging from slurry packing, electrokinetic, centripetal, andsupercritical fluid packing methods. These all involve a plurality ofsteps to effect the packing process and even with close monitoring donot give as uniform a bed as desired for many applications.

Furthermore, a great degree of difficulty still remains associated withthe ability to pack long, narrow bore capillaries. In addition, mostpacked capillaries require end frits of a different material to retainthe packing particles within the packed capillary bed. Creation of thosefrits remains to be a problem in column preparation as these frits mustbe rigid enough to retain the packing particles under a wide range ofcolumn packing, rinsing and operating conditions. Yet these frits mustalso possess a highly porous structure to permit a uniform mobile phaseflow through the entire cross-section of the column. A further problemarises in that the presence of the frit material makes the packing inthe column non-homogeneous due to the presence of a different materialand this can cause problems with the separation characteristics of thefinal column.

Monolithic column technology can effectively overcome both of thedifficulties associated with conventional packed capillary columntechnology. In the monolithic approach, a continuous separation bed iscreated inside the capillary tube using a solution, which undergoes bothchemical and physical changes in the capillary environment to producethe separation bed. In addition, the choice of appropriate chemistryallows the porous bed to chemically bond to the inner walls of thecapillary by a condensation reaction and the resulting packed tube isalso homogeneous in nature.

The use of monolithic columns has been reported in gas and liquidchromatography and is also currently being used in CEC to alleviate theextensive labor involved with packed column fabrication. Moreover, thegreatest inherent advantage of the monolithic capillary columns is theelimination of the need for the end frits. The elimination of these endfrits allows the entire column to remain homogeneous, rather thanexhibiting different properties by the packing particles and the endfrits. It has also been demonstrated that the end frits reduce thecolumn's separation efficiency and are responsible for bubble formationduring the analysis.

Although much simpler than particle packed capillaries, monolithiccolumns derived by organic polymerization also possess certainlimitations. One critical drawback associated with this type ofmonolithic capillary is the tendency of the polymer network to swellduring exposure to certain organic solvents, which are contained in therunning mobile phase. This swelling may result in reductions in thepermeability of the monolith as a result of alterations in the porosityof the monolith. Such structural change ultimately leads to changes inthe column performance during the course of its use.

Unlike the monolithic separation beds from organic polymers, columnscontaining a porous silica-based monolithic matrix prepared throughsol-gel chemistry do not suffer from the swelling phenomena thusoffering a versatile and promising alternative to organic packedcapillaries. In addition, monolithic columns, since they are preparedwithout end frits can produce a homogeneous separation column, which ishighly desirable for a wide variety of separation techniques.

Pretorius was one of the first influential pioneers of CEC who, in 1974,demonstrated the advantages of electroosmosis as a pumping mechanism forchromatographic separations. Jorgenson and Lukacs published CEC analysesof 9-methylanthracene and perylene on an ODS-packed capillary column.Meanwhile, a 1987 report by Tsuda demonstrated the possibility ofachieving CEC separations by the simultaneous use of both electroosmoticand pressure-driven flows in the separation column. Yet Knox and Grantmade another significant contribution to the development of thistechnique Following this publication, the term “electrochromatography”became generally accepted and numerous researchers refocused theirattention to CEC.

As described earlier, two types of monolithic columns have beendeveloped: (1) organic polymer-based and (2) bonded silica-based. In thefirst approach, fabrication of a monolithic capillary column isaccomplished by polymerization reaction of organic monomericprecursor(s). Hileman et at used Carbowax coated open pore polyurethanemonolithic capillaries for the separations of several classes ofanalytes including aromatic hydrocarbons, aliphatic alcohols and metalchelates through gas chromatography. Hjerten et al prepared monolithiccapillaries with compressed polyacrylamide gels for separation ofproteins using HPLC and of low molecular mass compounds and basicproteins using CEC. Frechet and coworkers reported a series ofpublications on the use of methacrylate monomers for the preparation ofHPLC and CEC monolithic capillaries through copolymerization. Palm andNovotny prepared CEC monoliths using mixtures ofpolyacrylamide/polyethylene glycol, derived with either C₄ or C₁₂ligands, which were used to separate alkyl phenones and peptides.Additionally, Fujimoto et al reported the usage of cross-linkedpolyacrylamides for the separation of small dansylated amino acids andneutral steroids on monolithic CEC capillaries.

An alternative to a column with an organic polymer-based stationaryphase column is one with a bonded silica stationary phase prepared bysol-gel chemistry. Cortes and coworkers prepared porous beds bypolymerizing potassium silicate solutions in situ. The columnscontaining the porous beds were then packed with 5 μm Spherisorb ODSparticles for use in LC. Fields used solutions of potassium silicate andformamide to create a porous bed that was further reacted withdimethyloctadecylchlorosilane, and achieved plate heights of 65 μm inLC.

Tanaka and coworkers used the sol-gel technique for the development ofan octadecylsilylated, porous monolithic column for use in LC. In thisstudy, poly(ethylene oxide), PEO, was incorporated into a mixture oftetramethoxysilane (TMOS) and acetic acid to develop porous silica rods,followed by an in-column octadecylsilylation reaction. Followingwashings, and drying at 50° C. for three days, the silica rods were thentreated for two hours at 600° C.

Dulay et al used sol-gel technology for the preparation of monolithiccolumns loaded with 3 μm ODS particles. The sol-gel solution served as aretaining matrix immobilizing and shielding the ODS stationary phaseparticles. Sol-gel capillary columns containing the ODS embeddedparticles yielded CEC separation efficiencies on the order of 80,000plates/m (16,000 plates/column) for a test mixture of six unchargedpolyaromatic hydrocarbons or PAHs.

Lee and coworkers also used sol-gel chemistry to glue 7 μm ODS particlesthereby creating a continuous large-pore CEC column. The sol-geltechnology in this approach was used to create a bridge between adjacentparticles, as well as the capillary wall and particles in its vicinity,thereby eliminating the need for retaining end-frits, thus result beingefficient separations of small organic and aromatic amine compounds onsuch “sol-gel-glued” monolithic columns.

SUMMARY OF THE INVENTION

It is, therefore, an object of the invention to create a monolithiccolumn from a sol-gel process.

It is also an object of the invention to prepare a sol-gel column havinga porous, separation bed without the use of particles being incorporatedinto the bed.

It is a further object of the invention to create a sol-gel column in asingle-step process that obviates the need for a plurality of processingsteps.

It is another object of the invention to produce a monolithic sol-gelcolumn, which is chemically bonded to the capillary wall.

It is a further object of the invention to produce a monolithic sol-gelcolumn that does not require high temperature processing steps.

It is another object of the invention to produce a monolithic sol-gelcolumn having an optical window useful for on-column detection studiesof the analytes separated by the separation column, using variousspectral techniques.

BRIEF DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention are readily appreciated as thesame becomes better understood by reference to the following detaileddescription when considered in connection with the accompanying drawingswherein:

FIG. 1 is a scanning electron micrograph of a sol-gel monolithic column,cross-sectional view, magnified 1,800 times;

FIG. 2 is a scanning electron micrograph of a sol-gel monolithic column,longitudinal view, magnified 7,000 times;

FIG. 3 is a scanning electron micrograph of a sol-gel monolithic column,longitudinal view, magnified 15,000 times;

FIG. 4 is a graph of the effect of the change of electroosmotic mobilitywith an increase in percentage of acetonitrile and Tris-HCl in a mobilephase;

FIG. 5 represents the CEC analysis of a mixture of PAHs on a 50 cm×50 μlODS sol-gel monolithic column;

FIG. 6 shows plate height versus flow rate within a sol-gel mediated ODSmonolithic capillary;

FIG. 7 is a separation analysis of a mixture of benzene derivatives on asol-gel mediated ODS monolithic column;

FIG. 8 is a CEC separation of a mixture of aldehydes and ketones on asol-gel mediated ODS monolithic column; and

FIG. 9 is a schematic showing the various steps in preparing amonolithic separation column having an additional optical window in thestructure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the present invention, monolithic sol-gel columns are prepared by anin situ creation of chromatographic stationary phases withsurface-bonded ligands. Unlike conventional techniques, various columnpreparation processes, such as deactivation, coating/packing,stationary-phase immobilization and end frit making, are carried out inone single step, thus reducing the time and labor associated with columnfabrication. In addition, the process produces a column that ishomogenous since there are no particles included in the sol-gel and thesol-gel monolithic bed actually forms bonds with the fused silicacapillary surface, making a unitary structure across the diameter of thetube.

In order to achieve the desired sol-gels of the instant invention,certain reagents in a reagent system were preferred for the fabricationof the gels for the monolithic columns of the present invention. Thereagent system included two sol-gel precursors, a deactivation reagent,one or more solvents and a catalyst. For the purposes of this invention,one of the sol-gel precursors contains a chromatographically activemoiety selected from the group consisting of octadecyl, octyl,cyanopropyl, diol, biphenyl, phenyl, cyclodextrins, crown ethers andother moieties. Representative precursors include, but are not limitedto: Tetramethoxysilane,3-(N-styrlmethyl-2-aminoethylamino)-propyltrimethoxysilanehydrochloride, N-tetradecyldimethyl(3-trimethoxysilylpropyl)ammoniumchloride, N(3-trimethoxysilylpropyl)-N-methyl-N,N-diallylammoniumchloride, N-trimethoxysilylpropyltri-N-butylammonium bromide,N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride,Trimethoxysilylpropylthiouronium chloride,3-[2-N-benzyaminoethylaminopropyl]trimethoxysilane hydrochloride,1,4-Bis(hydroxydimethylsilyl)benzene,Bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane,1,4-bis(trimethoxysilylethyl)benzene, 2-Cyanoethyltrimethoxysilane,2-Cyanoethyltriethoxysilane, (Cyanomethylphenethyl)trimethoxysilane,(Cyanomethylphenethyl)triethoxysilane,3-Cyanopropyldimethylmethoxysilane, 3-Cyanopropyltriethoxysilane,3-Cyanopropyltrimethoxysilane, n-Octadecyltrimethoxysilane,n-Octadecyldimethylmethoxysilane, Methyl-n-Octadecyldiethoxysilane,Methyl-n-Octadecyldimethoxysilane, n-Octadecyltriethoxysilane,n-Dodecyltriethoxysilane, n-Dodecyltrimethoxysilane,n-Octyltriethyoxysilane, n-Octyltrimethoxysilane,n-Ocyldiisobutylmethoxysilane, n-Octylmethyldimethoxysilane,n-Hexyltriethoxysilane, n-isobutyltriethoxysilane,n-Propyltrimethoxysilane, Phenethyltrimethoxysilane,N-Phenylaminopropyltrimethoxysilane, Styrylethyltrimethoxysilane,3-(2,2,6,6-tetramethylpiperidine-4-oxy)-propyltriethoxysilane,N-(3-triethoxysilylpropyl)acetyl-glycinamide,(3,3,3-trifluoropropyl)trimethoxysilane, and(3,3,3-trifluoropropyl)methyldimethoxysilane.

A second sol-gel precursor,N-Octadecyldimethyl[3-(trimethoxysilyl)propyl]ammonium chloride, wasfound to be critical since it possessed an octadecyl moiety that allowedfor chromatographic interactions of analytes with the monolithicstationary phase. Additionally, this reagent served to yield apositively charged surface thereby providing the relatively highelectroosmotic flow necessary in capillary electrochromatography.However, it is considered within the scope to use any other reagent asknown to one of ordinary skill in the art that would contain theoctadecyl moiety for the purposes already set forth.

The deactivation reagent, Phenyldimethylsilane, and the catalyst,Trifluoroacetic acid, were selected for the preparation of the columnsof the instant invention, however, any know deactivation reagent and/orcatalyst as known to those of ordinary skill in the art may be used.

The sol-gel solutions were prepared by mixing 100 μL ofTetramethyloxysilane (TMOS) with 100 μL of C₁₈-TMS(N-Octyldecyl-dimethyl[3-(trimethoxy-silyl)propyl]ammonium chloride), 10μL of PheDMS (Phenyldimethylsilane), 100 μL of 99% Trifluoroacetic acid(TFA) (containing 10% water), and 100 μL of 90% TFA (containing 10%water) in a micro vial. This mixture was thoroughly vortexed for 5minutes, and the precipitate was then separated from the sol-gelsolution through centrifugation at 13,000 rpm for 5 minutes. Thesupernatant was decanted into another micro vial and used for thecreation of the monolithic separation bed.

A standard fused silica capillary tube, as known to those of skill inthe art, was selected to be filled with the sol-gel solution. The tubeused here was externally coated with polyimide, however it is within thescope of the invention to use a tube coated with any polymer or othercoating such as metal, as known in the art; the external coating servingas a structural integrity device for the tube.

Prior to filling with the sol-gel solution, the inner surface of thecapillary was first treated with deionized water. For this, anapproximately 5 meter long section of 50 μm internal diameter fusedsilica capillary was rinsed with deionized water for approximately 15minutes under a helium pressure of 200 psi. The capillary was thenemptied by expelling the water from within by using the same heliumpressure. Both ends of the capillary tube were then fused using anoxyacetylene torch, and the capillary was placed in a GC oven forthermal conditioning by raising the temperature at 0.5° C./min from 40°C. to a final temperature of 250° C. with a hold time of 60 minutes at250° C. The column was then removed from the GC oven, and the ends wereopened, followed by purging of the column with helium under 200 psipressure for an additional 30 minutes.

Next, a desired length, for example 60 cm, of the hydrothermallypretreated fused-silica capillary was taken and installed into thecapillary filling chamber, containing a polyethylene microcentrifugevial with the desired sol-gel solution. It is, of course, within thescope of the invention to use any desired length as desired by one ofskill in the art. Using 100 psi helium pressure, the sol solution waspushed into the column. The column, containing the sol solution, wasthen allowed to remain installed in the pressurized capillary chamberand left undisturbed for approximately four hours until gelation of thesol solution was visually apparent. Following this, the pressure wasslowly released and the column was removed from the capillaryfilling/purging chamber. It was then affixed perpendicular to the benchtop. A 60-s epoxy seal was then applied to the ends of the capillary toensure adequate sealing prior to its thermal conditioning. Next, a veryslow thermal conditioning program was used. An example of this thermalconditioning consists of a programmed temperature heating at 0.2° C./minfrom 35° C. (1 minute hold time) to a final temperature of 150° C.,where the column has held for 120 minutes. Following heating, the endswere cut open and the monolithic capillary was then installed into aBio-Rad CE system for subsequent rinsing at 100 psi. It is, of course,contemplated that any CE system as known to those of skill in the artmay also be used. The monolithic column was initially rinsed with 100%HPLC grade acetonitrile, followed by a 50:50 acetonitrile/deionizedwater solution for periods of 5 minutes each, and finally the desiredrunning mobile phase for 15 minutes prior to conducting columnevaluation and/or analysis.

Visualization of the monolithic microstructure within the capillary tubewas accomplished through the use of a scanning electron microscope. Allscanning electron micrographic images were acquired from sections of themonolithic column initially cut into equal lengths, those beingapproximately 2.5 mm, and positioned perpendicularly within aretractable aluminum stage using a double-sided tape. These samples werethen used to obtain cross-sectional views of the monolithic CEC columns.Longitudinal sections were acquired by dissecting approximately 1.0 cmsections of capillary at approximately 45°, thus yielding a capillarysegment revealing a protruding portion of the monolithic matrix withoutthe top portion of the fused silica present. These sections were thenmounted parallel on an aluminum stage with the aid of double-sidedcarbon tape. Both stages, with all mounted capillary segments, were thenconsecutively placed into a Balzers SCD 050 sputter coating chamber andcoated with a gold/palladium alloy at 40 mA for 60 seconds to avertsubsequent charging.

FIG. 1 represents a SEM cross-sectional view of the sol-gel monolithiccolumn at a magnification of 1800×. Observations at this magnificationreveal that the entire cross-section of the capillary contains themonolithic matrix. FIG. 2, a longitudinal view of the monolithiccapillary at 7000× magnification, reveals the porous structure of themonolithic matrix. From this view it is evident that the pore diametersare of approximately 1.5 μm. The use of higher C₁₈TMS-to-TMOS molarratios in the sol solution provided monolithic beds with the said porecharacteristics. This also allowed for enhanced permeability of themobile phase.

For example, an increase in the C₁₈TMS-to-TMOS molar ratio of from 0.5to 0.75 yielded flow rates of up to approximately 7.75 μL/min for themobile phase consisting of 80% (v/v) Acrylonitrile 20% (v/v)/5 mMTris-HCl. Dimethylsulfoxide (DMSO) was used as the neutral EOF marker todetermine the linear velocity of the mobile phase and was found to be0.97 mm/s using the mobile phase.

FIG. 3, a cross-sectional view of the inner capillary at highermagnification (15000×), reveals the chemical bonding during the columnpreparation process that occurred due to the condensation between thesol-gel network structure and the silanol moieties on the innercapillary walls.

The scanning electron micrograph studies show that sol-gel chemistryprovides a unique, yet simple mechanism for the fabrication of CECmonolithic columns. One of the key sol-gel reactions consists of thehydrolysis of the precursors. This is shown below with respect to theuse of TMOS and C₁₈TMS. It is understood that this choice of reagents isfor illustrative purposes only and others can be used as describedbefore:

The Complete Hydrolysis ofN-Octadecyldimethyl[3-(thimethyloxysilyl)propyl]ammonium chlorideC₁₈-TMS (a) and tetramethoxysilane (TMOS) (b)

As shown above, the nucleophilic attack of water molecules on thesilicon atom results in the replacement of the methoxy substituents withhydroxy moieties. As the sol-gel reactions proceed, the products of thehydrolysis can then undergo polycondensation reactions in a variety ofways: (a) between hydrolyzed products of the same original precursor,(b) between hydrolyzed products of two different original precursors,and (c) between the hydrolyzed products of either precursor with thesilanol groups on the inner capillary surface. A simplifiedrepresentation of a polycondensation reaction between the hydrolysisproducts of both precursors is depicted below:

Condensation of Tetrahydroxysilane withN-Octadecyldimethyl[3-(trihydroxysilyl)propyl]ammonium chloride

This growing three-dimensional polymeric network will then eventuallybecome anchored to the inner capillary surface through chemical bondingwith the silanol moieties residing along the inner fused-silicacapillary surface. This is shown by the following:

Condensation of the Fused-Silica Surface with the Growing Sol-GelNetwork Containing Chemically Bonded Residue ofN-Octadecyldimethyl[3-(trihydroxysilyl)propyl]ammonium chloride

Finally, the incorporation of the PheDMS into the sol solution serves asa deactivating reagent for the monolithic bed. This deactivation reagentis initially added to the sol solution. The mobile hydrogen bonded tosilicon atom in the structure of this reagent is reactive toward silanolgroups, especially at elevated temperatures. It can be assumed thatduring the sol-gel process, this reagent becomes physically incorporatedin the monolithic structure but subsequently, during thermal treatmentof the column, reacts with the residual silanol groups in the monolithicstructure providing deactivation, as shown below:

Deactivation of the Sol-Gel ODS Monolith with Phenyldimethylsilane

Thus it has been shown that the sol-gel process provides a chemicalanchorage of the monolithic matrix to the inner walls of the capillary(FIG. 3). This thoroughly illustrates a significant attribute of thesol-gel monolithic column. Another aspect is that the sol-gel monolithicbed is completely held in the column chemical bonding with the walls ofthe capillary, thus obviating the need for any end frits to hold thecolumn material in place within the capillary.

Several analyses were performed using the monolithic columns of theinstant invention. Each mobile phase was prepared by mixing the desiredvolumes of acetonitrile with a Tris-HCl background electrolyte solution.The organic solvent and the background electrolyte solution werethoroughly degassed individually via simultaneous ultrasonication andhelium purging for approximately one hour prior to mixing and usage.Thorough degassing of the mobile phase was necessary to preventsubsequent bubble formation/generation during usage. This initialdegassing procedure allowed for electrochromatographic experiments to becontinuously performed without pressurization of the mobile phase. Toachieve the desired concentration of aqueous electrolyte, a 50 mMsolution was initially prepared followed by dilution to achieve the 5 mMconcentration. The pH of this 5 mM solution was then measured andadjusted to approximately 2.3 by using concentrated HCl. This 5 mMTris-HCl, having a pH of approximately 2.3 solution, in conjunction with100% acetonitrile was individually degassed by simultaneousultrasonication and helium purging, followed by mixing the solution inappropriate volume ratios (e.g., 75% acetonitrile/25% 5 mM Tris-HCl,etc.) to prepare the running mobile phase.

Experiments were conducted for the investigation of the electroosmoticflow (EOF) in sol-gel monolithic columns. The first measurementsobtained using the monolithic ODS capillary was an evaluation of theeffect of acetonitrile percentage in the running mobile phase on theelectroosmotic mobility. For this, a set of mobile phases containingvarying percentages of acetonitrile and 5 mM aqueous Tris-HCl wasutilized. In addition, DMSO was used as the neutral electroosmotic flowmarker. The results obtained from these experiments are depicted in FIG.4. As illustrated, the electroosmotic mobility within the ODS monolithiccapillary consistently increased with the acetonitrile content in themobile phase. Such an increase in EOF is indicative of an increase inthe net positive surface charge within the monolithic columns. Onepossibility for this to occur is the reduction of effective negativesurface charge due to an increase in acetonitrile concentration in themobile phase, resulting in an equivalent increase of the effectivepositive surface charge due to the quaternary ammonium groups. This ispossible because the negative charge on the monolith/capillary isreduced due to the interaction of acetonitrile with the negative-chargegenerating surface groups such as the silanols.

In CEC, a consistent EOF is essential to drive the analtye(s) throughthe separation column. This EOF is generated due to an electrical doublelayer at the interface of the solid support with the liquid mobilephase. Most commonly, silica is used as the solid support and develops anegative surface charge under CE/CEC running conditions, presumably as aresult of the deprotonation of the silanol groups. The negativelycharged substrate attracts cations from the electrolyte in the mobilephase thereby forming the electrical double layer.

In this invention, the positively charged quaternary ammonium moietycontained in the N-octadecyldimethyl[3-(trimethoxysilyl)propyl]ammoniumchloride provided a positively charged surface on the monolithic matrix,which, in turn, counteracted on the effects of the residual silanolgroups residing both on the monolith and on the inner capillary surface.Under the experimental conditions used, a strong EOF was observed in thereversed direction (from cathode to anode), suggesting that the surfacepositive charge due to quaternary ammonium functionality in thesurface-bonded C₁₈TMS moieties is the EOF-determining factor in theprepared sol-gel monolithic columns.

The CEC analysis of a mixture of PAHs on a sol-gel ODS monolithic columnis shown in FIG. 5. A separation column 50 cm×50 μm inner diameter (46.1cm effective length) is used. The separation conditions were as follows:

Injection −12 kV for 0.03 min Run −15 kV, 2.68 μA Mobile phase 80%acrylonitrile/20% 5 mM Tris-HCl, pH 2.34, DMSO used as the EOF markerAnalytes (1) benzene 4.4053 × 10⁻⁶ M (2) naphthalene 2.7087 × 10⁻⁶ M (3)impurity (4) fluorene 1.5433 × 10⁻⁶ M (5) phenanthrene 1.5748 × 10⁻⁶ M(6) anthracene 9.6850 × 10⁻⁷ M (7) fluoranthene 1.1654 × 10⁻⁶ M (8)pyrene 1.2283 × 10⁻⁶ M (9) benzo[a]pyrene 1.5118 × 10⁻⁶ MThe monolithic sol-gel column allowed for the use of a mobile phasecontaining a higher percentage of acetonitrile (up to 80%) andsimultaneously rendered sufficient solute-stationary-phase interactions.The separation efficiencies acquired for naphthalene in the mixture ofPAH analytes in this analysis were on the order of 145,800 theoreticalplates per meter (73,000 plates/column). Because monolithic columns withoverall lengths of up to several meters can be easily prepared by thepresented sol-gel technology and that the prepared columns can beoperated using commercially available CE instrumentation, newpossibilities for generating extremely high efficiencies per column inCEC separations are created.

Van Deemer plots, as depicted in FIG. 6, were constructed throughvariations in the operating voltages, thereby altering the mobile-phaseflow rate through the column and measuring the achieved plate heightscorresponding to each operating voltage. The conditions were as follows:

Injection −12 kV for 3 sec Run −3 to −19 kV Mobile phase 75%acetonitrile/25% 5 mM Tris-HCl, pH 2.34, DMSO used as the EOF markerTest solutes (a) naphthalene (b) anthraceneFor the used test solutes, the Van Deemer plots reveal minimal increasesin plate heights as the mobile-phase flow rates are enhanced. Therelatively flat right-hand portion of the H vs u curves indicate anefficient mass-transfer process between the mobile phase and themonolithic ODS separation bed.

As can be seen in FIG. 6, the optimum linear velocity for the usedsol-gel monolithic ODS column was 0.75 mm/s, which corresponds toapplied field strength of −240 V/cm (−12 kV) in the sol-gel monolithiccolumns. This shows a new possibility for use of longer sol-gel columnsthat produce higher overall column efficiencies without exceeding theupper voltage limits of commercially available CE instruments.Furthermore, the use of sol-gel technology to prepare these monolithicODS columns for CEC is further accentuated as increased column lengthscan be used because the highly porous structure of the monoliths allowsfor their rinsing and CEC operation using commercially available CEinstrumentation without any additional pressurization capability. Therewas no need for pressurization of both capillary ends during analysis orfor increased pressurization for capillary rinsing prior to analysis. Nobubble formation was detected during analysis with the monolithiccapillaries when using electric field strengths of up to 300 v/cm. Inaddition, the highly porous structure of the monolithic capillariesallowed for operation without the need for modification to thecommercial CE instrument.

A test mixture of benzene derivatives was also used to further evaluatethe separation performance of the sol-gel ODS monolithic columns using amobile phase containing 75% acetonitrile and 25% aqueous 5 mM Tris-HClat pH of 2.34. Column efficiencies on the order of 163,200 plates/m(81,600 plates/column) were obtained in these analyses as shown in FIG.7.

FIG. 8 illustrates an analogous separation of a mixture of aldehydes andketones obtained on a sol-gel monolithic ODS column. As in the case withthe benzene derivatives, this probe mixture contained more closelyrelated analytes. Column efficiencies on the order of 174,600 plates/m(87,300 plates/column) were obtained in these analyses. The conditionshere were:

Separation column 50 cm × 50 μm (inner diameter) (46.1 cm of effectivelength) Injection −12 kV for 0.03 min −25 kV 0.5 μA run Mobile phase 70%acrylonitrile/30% Tris-HCl, pH 2.34 Analytes (a) benzaldehyde, 1.180 ×10⁻³ M (b) o-tolualdehyde, 1.9655 × 10⁻³ M (c) butyrophenone, 3.2680 ×10⁻⁴ M (d) valerophenone, 1.5263 × 10⁻⁴ M (e) hexaphenone, 3.0618 × 10⁻⁴M (f) heptaphenone, 2.986 × 10⁻⁴ M.

Repeatability studies were performed using various analyte mixtures.These experiments were essential to evaluate the consistency in soluteretention on the sol-gel monolithic ODS columns. The following tablepresents the CEC characteristics of sol-gel monolithic columns andexperimental data on retention time repeatability for a test mixture ofseven aromatic aldehydes and ketones.

Separation Effici- Reten- Separa- ency, N tion tion (plates/ t_(R)factor, Factor, R.S.D. Analyte column) (min) k α s (n = 5) Benzaldehyde89 778 9.144 0.050 0.027 0.295% Tolualdehyde :91 039  9.526 0.094 0.3820.029 0.302% Butyrophenone 83 867 10.048 0.154 0.522 0.025 0.248%Valerophenone 79 353 10.678 0.227 0.630 0.016 0.154% Hexaphenone 86 02711.550 0.327 0.872 0.022 0.194% Heptaphenone 89 687 12.788 0.469 1.2380.031 0.244%As depicted in this table, consistent repeatability values areexemplified by the low RSD (0.15-0.30%) values for solute retentiontimes in a series of five consecutive runs.

An additional embodiment of the invention is shown in FIG. 9. Thisembodiment provides an optical window that allows for the use of adetection device to monitor the eluted analytes after passing throughthe separation bed. Such an optical window allows for detection orfurther analysis of samples.

More specifically, the optical window is formed in an area or segmentcontaining gas 10 of the capillary tube, as generally shown at 12,adjacent the sol-gel bed 14. Normally, to provide structural integrityand strength to the tube, an outer coating 16 is provided around theentire outer surface of the tube 12. This outer coating 16 can be in theform of a polymer, metal or other coating as known in the art. Inaccordance with this embodiment of the present invention, this outercoating 16 is removed from the tube adjacent the sol-gel bed 14, asshown by 18, defining an optical window 30 in the tube in the area ofthe gas containing area 10.

Generally, the capillary tube 12 itself is optically transparent.However, the outer protective coating 16 interferes with the opticalproperties thereof. In order to provide the optical window 30, one endof the tube is sealed, as in 20. Under pressure, the sol-gel solution isintroduced, thereby compressing the gas within the capillary tube innergas containing area 10. This forces the formation of a compressed gasspace 24, separating an end portion of the bed 14 from the sealed endportion 20. A portion of the outer coating is removed, by burning or byother means known in the art, in the area of the gas containing area 10,thus forming the optical window 30, in the capillary tube. This windowmay be of any desired size known to those of ordinary skill in the art.

In this embodiment, the separation bed may also be thermally and/orsolvent treated as described earlier, and any further necessarypretreatment steps prior to use of the column is considered here asbeing within the scope of the invention.

A preferred method for forming this window involves the following steps:

-   -   (a) selection of an externally coated optically transparent        capillary tube, such as one containing a fused-silica inner        surface, the coating being any as known to those of ordinary        skill in the art, such as polymer, metal or other removable        coatings;    -   (b) sealing of the distal capillary end;    -   (c) filling the sealed capillary with sol-gel solution, the        pressure of the compressed gas in the sealed portion forming a        gas pocket in the distal end thereof;    -   (d) allowing the sol-gel solution inside the capillary to        transform into a porous monolithic bed;    -   (e) sealing the proximal end of the capillary tube to allow        thermal conditioning;    -   (f) removing the seals at both ends of the filled capillary tube        to allow for solvent conditioning; and    -   (g) removing a portion of the outer coating of the capillary        tube at the distal end in the region of the former gas pocket to        provide a window of any desired length for optical spectrometric        analyses.

The incorporation of the optical window allows for additional analysesto be performed on the sample as it exits the separation bed. The choiceof spectrometer is within the scope of ordinary skill in the analyticalchemistry art, and any known instrument is considered within the scopeof the invention. It is also understood that any form of pretreatment ofthe column may be used including thermal and solvent or both, but thechoice of pretreatments is solely a matter of choice.

It may be appreciated by one skilled in the art that additionalembodiments may be contemplated, including alternate reagents and tubesused for the outer matrix for the sol-gel filling.

In the foregoing description, certain terms have been used for brevity,clarity and understanding, but no necessary limitations are to beimplied therefrom beyond the requirements of the prior art, because suchwords are used for description purposes herein and are intended to bebroadly construed. Moreover, the embodiments of the apparatus andreagents used herein are by way of example, and the scope of theinvention is not limited to those in either construction or chemistry.

Having now described the invention, the preferred embodiments thereofand the advantageous new and useful results obtained thereby, along withreasonable chemical equivalents thereof as obvious to those of ordinaryskill in the art, these are now set forth in the appended claims.

REFERENCES

-   Behnke, B.; Bayer, E., J. Chromatogr. A, 1994, 680, 93.-   Behnke, B.; Grom, E.; Bayer, E., J. Chromatogr. A, 1995, 716, 207.-   Brinker, C. J.; Scherer, G. W., Sol-gel Science: The Physics and    Chemistry of Sol-gel Processing: Academic Press, San Diego, Calif.    1990.-   Chong, S.; Wang, D.; Hayes, J. D.; Malik, A. Anal. Chem. 1997, 69,    3889.-   Colon, L. A.; Fermier, A. M.; Guo, Y.; Reynolds, K. J., Ninth    International Symposium on High Performance Capillary    Electrophoresis (HPCE '97), Anaheim, Calif. 1997.-   Cortes, H. J.; Pfeiffer, C. D.; Richter, B. E.; Stevens, T. S., J.    High Resolut. Chromatogr. And Chromatogr. Commun., 1987, 10, 446.-   Dittmann, M. M.; Rozing, G. P., J. Chromatogr. A, 1996, 744, 63.-   Dulay, M. T.; Yan, C.; Rakestraw, D. J.; Zare, R. N., J. Chromatogr.    A, 1996, 725, 361.-   Dulay, M. T.; Kulkarni, R. P.; Zare, R. N., Anal. Chem., 1998, 70,    5103.-   Ericson, C.; Liao, J.; Nakazato, K.; Hjerten, S., J. Chromatogr. A    1997, 767, 33.-   Ericson, C.; Hjerten, S., Anal. Chem. 1999, 71, 1621.-   Fields, S. N., Anal. Chem., 1996, 68, 2709.-   Frame, L. A.; Robinson, M. L.; Lough, W. J., J. Chromatogr. A, 1998,    798, 243.-   Fujimoto, C.; Fujise, Y.; Matsuzawa, E., Anal. Chem., 1995, 716,    107.-   Fujimoto, C.; Fujise, Y.; Matsuzawa, E., Anal. Chem., 1996, 68,    2753.-   Hayes, J. D.; Malik, A., J. Chromatogr. B 1997, 695, 3.-   Hayes, J. D., Malik. A., manuscript in progress.-   Hileman, F. D.; Sievers, R. E.; Hess, G. G.; Ross, W. D., Anal.    Chem., 1973, 45, 1126.-   Hjerten, S.; Liao, J. L; Zhang, R., J. Chromatogr., 1989, 473, 273.-   Hjerten, S.; Li, Y. M.; Liao, J. L.; Mohammad, J.; Nakazato, K.;    Pettersson, G., Nature, 1992, 356, 810.-   Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Soga, N.; Tanaka, N., J.    Chromatogr. A, 1998, 797, 131.-   Jorgenson, J. W.; Lucas, K. D., J. Chromatogr. A, 1981, 218, 209.-   Knox, J. H.; Grant, I. H., Chromatographia, 1987, 24, 135.-   Li, Y. M.; Liao, J. L.; Nakazato, K.; Mohammad, J.; Terenius, L.;    Hjerten, S., Anal. Biochem. 1994, 223, 153.-   Ludtke, S.; Adam T.; Unger, K. K., J. Chromatogr. A, 1997, 786, 229.-   Malik, A.; Chong, S. in Pawliszyn, J. (ed.), “Applications of Solid    Phase Microextraction,” Royal Society of Chemistry, 1999, United    Kingdom, Ch. 6, pp. 73-91.-   Minakuchi, H.; Nakanishi, K.; Soga, N.; Ishizuka, N.; Tanaka, N.,    Anal. Chem., 1996, 68, 3498.-   Minakuchi, H.; Nakanishi, K.; Soga, N.; Ishizuka, N.; Tanaka, N., J.    Chromatogr. A. 1997, 8, 547.-   Minakuchi, H.; Nakanishi, K.; Soga, N.; Ishizuka, N.; Tanaka, N., J.    Chromatogr. A, 1998, 797, 121.-   Moffatt, F.; Cooper, P. A.; Jessop, K. M., Anal. Chem., 1999, 71,    1119.-   Nakanishi, K. K; Minakuchi, H.; Soga, N.; Tanaka, N., J. Sol-gel    Sci. and Tech. 1997, 762, 135.-   Palm, A.; Novotny, M. V., Anal. Chem. 1997, 69, 4499.-   Peters, E. C.; Petro, M.; Svec, F.; Frechet, J. M. J., Anal. Chem.    1997, 69, 3646.-   Peters, E. C.; Petro, M.; Svec, F.; Frechet, J. M. J., Anal. Chem.    1998, 70, 2288.-   Pietrzyk, D. J. in Packings and Stationary Phases in Chromatographic    Techniques; Unger, K. K., Ed.; Vol. 47; Marcel Dekker: New York    1990; Chapter 10.-   Pretorius, V; Hopkins, B. J.; Schieke, J. D., J. Chromatogr. A,    1974, 99, 23.-   Seifer, R. M.; Kraak, J. C.; Th. Kok, W.; Poppe, H., J. Chromatogr.    A, 1998, 808, 71.-   Smith, N. W.; Evans, M. C., Chromatographia 1994, 38, 649.-   Svec, F.; Frechet, J. M. J., Anal Chem. 1992, 64, 820.-   Svec, F.; Frechet, J. M. J., J. Chromatogr. A 1995, 702, 89.-   Tang, Q.; Wu, N.; Lee, M. L., J. Microcol. Sep., 1999, 11, 550.-   Tsuda, T., Anal. Chem., 1987, 59, 521.-   Van den Bosch, S. E.; Heemstra, S.; Kraak, J. C.; Poppe, H., J.    Chromatogr. A, 1996, 755, 165.-   Wang, Q. C.; Svec, F.; Frechet, J. M. J., Anal. Chem. 1993, 65,    2243.-   Wang, D.; Chong, S.; Malik, A. Anal. Chem. 1997, 69, 4566.-   Wei, W.; Luo, G. A.; Hua, G. Y.; Yan, C., J. Chromatogr. A, 1998,    817, 65.-   Xin, B.; Lee, M. L., Electrophoresis, 1999, 20, 67.-   Yan, C.; Dadoo, R.; Zhao, H.; Zare, R. N.; Rakestraw, D. J., Anal.    Chem., 1995, 67, 2026.-   Yang, C.; El Rassi, Z. Electrophoresis 1998, 19, 2278.-   Yang, C.; El Rassi, Z., J. Jiq. Chromatogr. 1995, 18, 3373.-   Zhang, M.; El Rassi, Z., Electrophoresis, 1998, 19, 2068.-   Zhang, M.; El Rassi, Z., Electrophoresis, 1999, 20, 31.-   Zhang, M.; Yang, C.; El Rassi, Z., Anal. Chem., 1999, 71, 3277.-   Zimina, T. M.; Smith, R. M.; Meyers, P., J. Chromatogr. A, 1997,    758, 191.

1. A monolithic sol-gel column comprising: a) a hydrothermallypretreated fused-silica capillary tube including an inner surface; andb) a porous matrix of substantially homogenous composition and beingfree of chromatographic particles, said porous matrix comprising i) afunctional group having a positive or negative charge; and ii) at leastone chromatographically active moiety chemically bonded to said porousmatrix; wherein said porous matrix is chemically bonded to said innersurface of said capillary tube; wherein said column comprises agas-filled portion distal to said porous matrix and that has not beencontacted by said porous matrix and that has smooth surfacecharacteristics and that is optically transparent, and wherein saidcolumn does not comprise an end frit; and wherein said porous matrix isformed in situ by a one-step process inside said capillary tube.
 2. Themonolithic sol-gel column according to claim 1, wherein said porousmatrix comprises:


3. The monolithic sol-gel column according to claim 1, wherein saidmatrix is formed using a sol-gel precursor that comprises a strong basicfunctional group or a strong acidic functional group.
 4. The monolithicsol-gel column according to claim 3, wherein said strong basicfunctional group is a quaternary amine group.
 5. The monolithic sol-gelcolumn according to claim 3, wherein said strong acidic functional groupis a sulfonic acid group.
 6. The monolithic sol-gel column according toclaim 1, wherein said chromatographically active moiety is selected fromthe group consisting of octadecyl, octyl, cyanopropyl, diol, biphenyl,phenyl, cyclodextrins, and crown ethers.
 7. The monolithic sol-gelcolumn according to claim 1, wherein said functional group provides apositive charge to said porous matrix.
 8. The monolithic sol-gel columnaccording to claim 1, wherein said functional group provides a negativecharge to said porous matrix.
 9. The monolithic sol-gel column accordingto claim 1, wherein said matrix is formed using a sol-gel precursormolecule that comprises both said functional group and saidchromatographically active moiety.
 10. The monolithic sol-gel columnaccording to claim 1, wherein said chemical bond is a covalent bond. 11.A monolithic sol-gel column comprising: a) an optically transparenthydrothermally pretreated fused-silica capillary tube; b) a porousmatrix of substantially homogenous composition and being free ofchromatographic particles, said porous matrix comprising i) a functionalgroup having a positive or negative charge; and ii) at least onechromatographically active moiety chemically bonded to said porousmatrix; wherein said porous matrix is chemically bonded to said innersurface of said capillary tube; and wherein said porous matrix is formedin situ by a one-step process inside said capillary tube; and c) anoptically transparent window means within a portion of said tube forproviding for optical analysis of analytes after said analytes travelthrough said porous matrix, wherein said optically transparent window isformed in an area of said capillary tube containing gas, adjacent tosaid porous matrix; and wherein said column does not comprise an endfrit.
 12. The monolithic sol-gel column according to claim 11, whereinan outer-coating is provided around the outer surface of said capillarytube.
 13. The monolithic sol-gel column according to claim 12, whereinsaid outer-coating is a polymer or a metal.
 14. The monolithic sol-gelcolumn according to claim 12, wherein a portion of said outer-coating isremoved from said capillary tube adjacent to said porous matrix therebydefining said optical window in the tube.
 15. The monolithic sol-gelcolumn according to claim 11, wherein said porous matrix comprises:


16. The monolithic sol-gel column according to claim 11, wherein saidporous matrix is formed using a sol-gel precursor that comprises astrong basic functional group or a strong acidic functional group. 17.The monolithic sol-gel column according to claim 16, wherein said strongbasic functional group is a quaternary amine group.
 18. The monolithicsol-gel column according to claim 16, wherein said strong acidicfunctional group is a sulfonic acid group.
 19. The monolithic sol-gelcolumn according to claim 11, wherein said chromatographically activemoiety is selected from the group consisting of octadecyl, octyl,cyanopropyl, diol, biphenyl, phenyl, cyclodextrins, and crown ethers.20. The monolithic sol-gel column according to claim 11, wherein saidfunctional group provides a positive charge to said porous matrix. 21.The monolithic sol-gel column according to claim 11, wherein saidfunctional group provides a negative charge to said porous matrix. 22.The monolithic sol-gel column according to claim 11, wherein said matrixis formed using a sol-gel precursor molecule that comprises both saidfunctional group and said chromatographically active moiety.
 23. Themonolithic sol-gel column according to claim 11, wherein said chemicalbond is a covalent bond.